Difference between revisions of "20.109(F18):Confirm gRNA sequence (Day5)"

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#*'''Please note:''' you will add one of your candidate clone plasmid mini-preps to each tube and the pdCas9 to both tubes!
 
#*'''Please note:''' you will add one of your candidate clone plasmid mini-preps to each tube and the pdCas9 to both tubes!
 
#Acquire an aliquot of the pdCas9 mini-prep (prepared by the teaching faculty) and of the competent MG1655 ''E. coli'' cells from the front laboratory bench.
 
#Acquire an aliquot of the pdCas9 mini-prep (prepared by the teaching faculty) and of the competent MG1655 ''E. coli'' cells from the front laboratory bench.
#Pipet 50 μL of the MG1655 competent cells into each labeled eppendorf tube.
+
#Pipet 100 μL of the MG1655 competent cells into each labeled eppendorf tube.
 
#*'''Remember:''' it is important to keep the competent cells on ice!  Also, avoid over pipetting and vortexing!
 
#*'''Remember:''' it is important to keep the competent cells on ice!  Also, avoid over pipetting and vortexing!
 
#Add 5 μL of the pdCas9 mini-prep to each tube.
 
#Add 5 μL of the pdCas9 mini-prep to each tube.

Latest revision as of 18:26, 25 October 2018

20.109(F18): Laboratory Fundamentals of Biological Engineering

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Fall 2018 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Measuring genomic instability        2. Modulating metabolism        3. Engineering biomaterials              


Introduction

Thus far in the module, your efforts have been focused on generating a gRNA molecule that will specifically target a gene in the E. coli fermentation pathway to increase either ethanol or acetate using the CRISPRi system. Today you will submit your pgRNA clones for sequencing analysis to confirm that your gRNA was indeed inserted and is correct. The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive process. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s and 2000s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (image on left, below). These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA’s sequence. In the “old days” radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image in center, below). More recently fluorescent dyes, one color linked to each dideoxy-base, have been used instead. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected (image on right, below). Your sample was sequenced in this way by Genewiz on an ABI 3730x1 DNA Analyzer.

Sequence trace data
Normal bases versus chain-terminating bases
Sequencing gel


In addition to the pgRNA plasmid you generated, the CRISPRi system requires a plasmid that contains the gene that encodes the dCas9 protein. The CRISPRi system is only active when both plasmids are present within a single cell. To ensure this is the case, each plasmid encodes a different antibiotic resistance cassette. As discussed previously in lecture, antibiotic cassettes are used to eliminate contamination and to be sure that the cells in your culture contain the plasmid(s) needed for your experiment. In our system, the pgRNA plasmid confers ampicillin resistance and the pdCas9 plasmid confers chloramphenicol resistance. For this reason we will use dual antibiotic selection conditions today and for the rest of our culture-based procedures in module 2.

Protocols

Part 1: Mini-prep pgRNA_target clones

The procedure for DNA isolation using small volumes is commonly termed "mini-prep," which distinguishes it from a “maxi-prep” that involves a larger volume of cells and additional steps of purification. The overall goal of each prep is the same -- to separate the plasmid DNA from the chromosomal DNA and cellular debris. In the traditional mini-prep protocol, the media is removed from the cells by centrifugation. The cells are resuspended in a solution that contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. A solution of sodium hydroxide and sodium dodecyl sulfate (SDS) is then added. The base denatures the DNA, both chromosomal and plasmid, while the detergent dissolves the cellular proteins and lipids. The pH of the solution is returned to neutral by adding a mixture of acetic acid and potassium acetate. At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. In addition, the DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The plasmid DNA renatures normally and stays in solution. Thus plasmid DNA got effectively separated from chromosomal DNA and proteins and lipids of the cell.

Today you will use a kit that relies on a column to collect the renatured plasmid DNA. The silica gel column interacts with the DNA while allowing contaminants to pass through the column. This interaction is aided by chaotropic salts and ethanol, which are added in the buffers. The ethanol dehydrates the DNA backbone allowing the chaotropic salts to form a salt bridge between the silica and the DNA.

  1. Pick up your two cultures, which are growing in test tubes labeled with your team color. Label two eppendorf tubes to reflect your samples (pgRNA_target#1 and #2).
  2. Vortex the bacteria and pour ~1.5 mL of each candidate into an eppendorf tube.
    Diagram showing how to aspirate the supernatant. Be careful to remove as few cells as possible.
  3. Balance the tubes in the microfuge, spin them at maximum speed for 2 min, and remove the supernatants with the vacuum aspirator.
  4. Pour another 1.5 mL of culture onto the pellet, and repeat the spin step. Repeat until you use up the entire volume of culture.
  5. Resuspend each cell pellet in 250 μL buffer P1.
    • Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
  6. Add 250 μL of buffer P2 to each tube, and mix by inversion until the suspension is homogeneous. About 4-6 inversions of the tube should suffice. You may incubate here for up to 5 minutes, but not more.
    • Buffer P2 contains sodium hydroxide for lysing.
  7. Add 350 μL buffer N3 to each tube, and mix immediately by inversion (4-10 times).
    • Buffer N3 contains acetic acid, which will cause the chromosomal DNA to messily precipitate; the faster you invert, the more homogeneous the precipitation will be.
    • Buffer N3 also contains a chaotropic salt in preparation for the silica column purification.
  8. Centrifuge for 10 minutes at maximum speed. Note that you will be saving the supernatant after this step.
    • Meanwhile, prepare 2 labeled QIAprep columns, one for each candidate clone, and 2 trimmed eppendorf tubes for the final elution step.
  9. Transfer the entire supernatant to the column and centrifuge for 1 min. Discard the eluant into a tube labeled 'Qiagen waste'.
  10. Add 0.5 mL PB to each column, then spin for 1 min and discard the eluant into the Qiagen waste tube.
  11. Next wash with 0.75 mL PE, with a 1 min spin step as usual. Discard the ethanol in the Qiagen waste tube.
  12. After removing the PE, spin the mostly dry column for 1 more minute.
    • It is important to remove all traces of ethanol, as they may interfere with subsequent work with the DNA.
  13. Transfer each column insert (blue) to the trimmed eppendorf tube you prepared (cut off lid).
  14. Add 30 μL of distilled H2O pH ~8 to the top center of the column, wait 1 min, and then spin 1 min to collect your DNA.
  15. Cap the trimmed tube or transfer elution to new eppendorf tube.
  16. Inform the teaching faculty when you have your purified plasmid so that the concentration of DNA can be measured.

Part 2: Transform CRIPSRi system into MG1655 E. coli cells

During “transformation,” a plasmid enters a bacterium and, once inside, replicates and expresses the genes it encodes. In our case, the goal is for each plasmid to enter a bacterium in a method referred to as co-transformation. As mentioned above, a gene on the pgRNA plasmid leads to ampicillin-resistance and a gene on the pdCas9 plasmid leads to chloramphenicol-resistance. Thus, only a co-transformed bacterium will grow on agar medium containing the antibiotics ampicillin and chloramphenicol. Untransformed cells will die before they can form a colony on the agar surface.

Bacterial transformation
Most bacteria do not usually exist in a “transformation ready” state, but the bacteria can be prepared such that they are permeable to the plasmid DNA. Cells that are capable of transformation are referred to as “competent.” Competent cells are extremely fragile and should be handled gently, specifically kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.

You will transform your pgRNA plasmids with the pdCas9 plasmid into E. coli MG1655, which is the strain we will use to examine the effect of your approach on either ethanol or acetate production.

  1. Label two 1.5 mL eppendorf tubes with your team information and clone designation (pgRNA#1 and pgRNA#2).
    • Please note: you will add one of your candidate clone plasmid mini-preps to each tube and the pdCas9 to both tubes!
  2. Acquire an aliquot of the pdCas9 mini-prep (prepared by the teaching faculty) and of the competent MG1655 E. coli cells from the front laboratory bench.
  3. Pipet 100 μL of the MG1655 competent cells into each labeled eppendorf tube.
    • Remember: it is important to keep the competent cells on ice! Also, avoid over pipetting and vortexing!
  4. Add 5 μL of the pdCas9 mini-prep to each tube.
  5. Add 5 μL of each pgRNA candidate clone mini-prep to the appropriate eppendorf tube.
  6. Incubate your co-transformation mixes on ice for 30 min.
  7. Carry your ice bucket with your co-transformations to the heat block at the front laboratory bench.
    • Be sure you also take your timer.
  8. Transfer the tubes with your co-transformations to the heat block set to 42 °C and incubate for exactly 45 sec.
  9. Remove your co-transformations from the heat block and immediately put them back in the ice bucket, then incubate for 2 min.
  10. Pipet 500 μL of pre-warmed SOC media into each co-transformation.
  11. Move your co-transformations to the 37 °C incubator and carefully place them on the nutator (secure your tubes by sliding them under the rubberband).
  12. Incubate co-transformations for 1 h.
  13. Retrieve your co-transformations from the incubator and alert the teaching faculty that you are ready to plate your samples.
  14. Plate 100μL of each co-transformation onto an appropriately labeled LB+Amp+Cam agar plate.
    • The teaching faculty will demonstrate how you should 'spread' your co-transformation onto the LB+Amp+Cam agar plates. You should include this procedure in your laboratory notebook.
  15. Move your spread plates to the 37 °C incubator wherein they will incubate for ~18 hr.

Part 3: Prepare pgRNA clones for sequencing analysis

Just as amplification reactions require a primer for initiation, primers are also needed for sequencing reactions. Legible readout of the gene typically begins about 40-50 bp downstream of the primer site, and continues for ~1000 bp at most. Thus, multiple primers must be used to fully view genes > 1 kb in size. Though the target sequence for your gRNA is shorter than 1000 bp, we will sequence with both a forward and reverse primer to double-check that the sequence is correct (i.e. free of unwanted mutations).

The primers you will use today are below:

Primer Sequence
gRNA confirmation forward primer 5' - GGG TTA TTG TCT CAT GAG CGG ATA CAT ATT TG - 3'
gRNA confirmation reverse primer 5' - CGC GGC CTT TTT ACG GTT C - 3'

Open the sequence document for pgRNA and create a Benchling file using the procedure outlined on M2D1. Label the file with the following features:

  • sequence that was added to the gRNA target oligo you designed on M2D2
  • sequence that is complementary to the universal CRIPSRi reverse primer (5' - ACT AGT ATT ATA CCT AGG ACT GAG CTA GC - 3')
  • sequence to which the gRNA confirmation forward and reverse primers anneal
  • ampicillin resistance cassette (1467-2126 bp)
  • ampicillin resistance cassette promoter (2366-2394 bp)

Using the sequences you highlighted, where do you think your gRNA target sequence was inserted? Create a new file that represents your pgRNA_target plasmid product.

The recommended composition of sequencing reactions is ~800 ng of plasmid DNA and 25 pmoles of sequencing primer in a final volume of 15 μL. The miniprep'd plasmid should have ~300 ng of nucleic acid/μL but that will be a mixture of RNA and DNA, so we will estimate the amount appropriate for our reactions.

Because you will examine the sequence of your potential plasmids using both a forward and a reverse primer, you will need to prepare two reactions for each candidate. Thus you will have a total of four sequencing reactions. For each reaction, combine the following reagents directly in the appropriate tube within the 8-PCR-tube strip, as noted in the table below:

  1. 6 μL nuclease-free water
  2. 4 μL of your plasmid DNA candidate
  3. 5 μL of the primer stock on the teaching bench (the stock concentration is 5 pmol/μL)
    • Please add the forward primer to the odd numbered tubes and the reverse primer to the even numbered tubes (i.e. tube #1 contains pgRNA#1 plasmid DNA and gRNA confirmation forward primer, tube #2 contains pgRNA#1 plasmid DNA and gRNA confirmation reverse primer, etc).

The side of each tube is numerically labeled and you should use only the four tubes assigned to your group. The teaching faculty will turn in the strips at the Genewiz company drop-off box for sequencing.

T/R Tubes W/F
Red 1-4 Yellow
Orange 5-8 Green
Green 9-12 Blue
Pink 13-16
Purple 17-20
21-24
25-28
29-32

Reagents

  • QIAprep Spin Miniprep Kit (Qiagen)
  • pdCas9 (concentration: 0.05 μg / μL )
  • Chemically competent E. coli MG1655 (genotype: F- lambda- ilvG- rfb-50 rph-1)
  • SOC medium
    • 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose
  • LB+Amp+Cam plates
    • Luria-Bertani (LB) broth contains 1% tryptone, 0.5% yeast extract, and 1% NaCl
    • Plates prepared by adding 1.5% agar, 100 μg/mL ampicillin (Amp), and 34 μg/mL chloramphenicol (Cam) to LB

Navigation links

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